Liposome-entrapped topoisomerase inhibitors

ABSTRACT

A composition for administration of a therapeutically effective dose of a topoisomerase inhibitor I or topoisomerase I/II inhibitor is described. The composition includes liposomes having an outer surface and an inner surface defining an aqueous liposome compartment, and being composed of a vesicle-forming lipid and of a vesicle-forming lipid derivatized with a hydrophilic polymer to form a coating of hydrophilic polymer chains on both the inner and outer surfaces of the liposomes. Entrapped in the liposomes is the topoisomerase inhibitor at a concentration of at least about 0.10 μmole drug per μmole lipid.

This application is a continuation of U.S. application Ser. No.10/230,796 filed Aug. 29, 2002, now pending which is a continuation ofU.S. application Ser. No. 10/046,326 filed Oct. 19, 2001, now U.S. Pat.No. 6,465,008; which is a continuation of U.S. application Ser. No.09/419,189 filed Oct. 15, 1999, now U.S. Pat. No. 6,355,268; whichclaims the benefit of U.S. Provisional Application No. 60/104,671, filedOct. 16, 1998, all of which are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to a liposome composition having anentrapped topoisomerase inhibitor.

BACKGROUND OF THE INVENTION

Next to heart disease, cancer is the major cause of death in the UnitedStates, causing over 500,000 fatalities annually (Katzung, B., “Basicand Clinical Pharmacology”, 7^(th) Edition, Appleton & Lange, StamfordConn., 1998, p. 882). With present methods of treatment, one-third ofpatients are cured with local measures, such as surgery or radiationtherapy, which are quite effective when the tumor has not metastasizedby the time of treatment. Earlier diagnosis might lead to increased cureof patients undergoing such local treatments. However, in many cases,early micrometastasis is a characteristic feature of the neoplasm,indicating that a systemic approach such as chemotherapy may berequired, often along with a local treatment method, for effectivecancer management.

Cancer chemotherapy can be curative in certain disseminated neoplasmsthat have undergone either gross or microscopic spread by the time ofdiagnosis. These include testicular cancer, diffuse large cell lymphoma,Hodgkin's disease and choriocarcinoma as well as childhood tumors suchas acute lymphoblastic leukemia. For other forms of disseminated cancer,chemotherapy provides a palliative rather than curative therapy.

Effective palliative therapy results in temporary clearing of thesymptoms and signs of cancer and prolongation of useful life.

Advances in cancer chemotherapy have recently provided evidence thatchemical control of neoplasia is possible for a number of cancers.

One category of drugs used for cancer therapy is topoisomeraseinhibitors. These compounds inhibit the action of topoisomerase enzymeswhich play a role in the replication, repair, genetic recombination andtranscription of DNA. An example of a topoisomerase inhibitor iscamptothecin, a natural compound that interferes with the activity oftopoisomerase I, an enzyme involved in DNA replication and RNAtranscription. Camptothecin and the camptothecin analogues topotecan andirinotecan are approved for clinical use.

Camptothecin and its analogues are effective in cancer chemotherapy byinterfering with the breakage/reunion actions of topoisomerase I. Thecompounds stabilize and form a reversible enzyme-camptothecin-DNAternary complex which prevents the reunion step of the breakage/unioncycle of the topoisomerase reaction.

One problem with camptothecin is its water insolubility, which hindersthe delivery of the drug. Numerous analogues of camptothecin have beenprepared to improve the compound's water solubility. Another problemwith camptothecin and its analogues is that the compounds aresusceptible in aqueous environments to hydrolysis at the α-hydroxylactone ring. The lactone ring opens to the carboxylate form of thedrug, a form that exhibits little activity against topoisomerase I.

Various approaches to improving the stability of camptothecin and itsanalogues have been described. One approach has been to entrap thecompounds in liposomes.

Burke (U.S. Pat. No. 5,552,156) describes a liposome compositionintended to overcome the instability of camptothecin and its analoguesby entrapping the compounds in liposomes having a lipid bilayer membranewhich allows the compound to penetrate, or intercalate, into the lipidbilayer. With the compound intercalated into the bilayer membrane, it isremoved from the aqueous environment in the core of the liposome andthereby protected from hydrolysis.

One problem with this approach is that the liposomes are quickly removedfrom the bloodstream by the reticuloendothelial system (RES), preventingdelivery, and preferably accumulation, at the tumor site.

Subramanian and Muller (Oncology Research, 7(9):461–469 (1995)) describea liposome formulation of topotecan and report that inliposome-entrapped form, topotecan is stabilized from inactivation byhydrolysis of the lactone ring. However, the biological activity of theliposome-entrapped drug in vitro has only 60% of the activity of thefree drug.

Lundberg (Anti-Cancer Drug Design, 13:453 (1998)) describes twolipophilic, oleic acid ester derivatives of camptothecin analogues whichare entrapped in liposomes and intercalated into the bilayer forstabilization of the lactone ring. Daoud (Anti-Cancer Drugs, 6:83–93(1995)) describes a liposome composition including camptothecin, wherethe drug is also intercalated into the lipid bilayer. The liposomes inboth of these references are prepared conventionally, where the drug ispassively entrapped in the liposomes to sequester the drug in the lipidbilayer membrane for stabilization. Using this method of preparation itis difficult to achieve a sufficient drug load in the liposomes forclinical efficacy.

Accordingly, there is still a need in the art for a liposome formulationwhich (i) includes a topoisomerase inhibitor, such as camptothecin andits analogues; (ii) remains in the bloodstream for a prolonged period oftime; (iii) retains antitumor activity; and (iv) includes a sufficientdrug load for clinical relevance.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide a topoisomeraseinhibitor composition for improved cancer therapy.

It is another object of the invention to provide a liposome compositionfor administration of a topoisomerase inhibitor for antitumor therapy.

In one aspect, the invention includes a composition for treating a tumorin a subject, comprising liposomes composed of a vesicle-forming lipidand between about 1–20 mole percent of a vesicle-forming lipidderivatized with a hydrophilic polymer. The liposomes are formed underconditions that distribute the polymer on both sides of the liposomes'bilayer membranes. Entrapped in the liposomes is a topoisomerase Iinhibitor or a topoisomerase I/II inhibitor at a concentration of atleast about 0.10 μmole drug per μmole lipid. The liposomes have aninside/outside ion gradient sufficient to retain the topoisomerase Iinhibitor or topoisomerase I/II inhibitor within the liposomes at thespecified concentration.

In one embodiment, the topoisomerase inhibitor is a topoisomerase Iinhibitor selected from the group consisting of camptothecin andcamptothecin derivatives. For example, the camptothecin derivative canbe 9-aminocamptothecin, 7-ethylcamptothecin, 10-hydroxycamptothecin,9-nitrocamptothecin, 10,11-methlyenedioxycamptothecin,9-amino-10,11-methylenedioxycamptothecin or9-chloro-10,11-methylenedioxycamptothecin. In other embodiments, thecamptothecin derivative is irinotecan, topotecan,(7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camptothecin,7-(4-methylpiperazinomethylene)-10,11-methylenedioxy-20(S)-camptothecinor 7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin.

In another embodiment, the topoisomerase inhibitor is a topoisomeraseI/II inhibitor, such as6-[[2-(dimethylamino)-ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-onedihydrochloride, azotoxin or3-methoxy-11H-pyrido[3′,4′-4,5]pyrrolo[3,2-c]quinoline-1,4-dione.

The hydrophilic polymer included in the liposome composition can bepolyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline,polyethyloxazoline, polyhydroxypropyloxazoline,polyhydroxypropylmethacrylamide, polymethacrylamide,polydimethylacrylamide, polyhydroxypropylmethacrylate,polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose,polyethyleneglycol and polyaspartamide.

In a preferred embodiment, the hydrophilic polymer is polyethyleneglycolhaving a molecular weight between 500–5,000 daltons.

In still another embodiment, the liposomes further include avesicle-forming lipid having a phase transition temperature above 37° C.

In yet another embodiment, the vesicle-forming lipid is hydrogenated soyphosphatidylcholine, distearoyl phosphatidylcholine or sphingomyelin.One preferred liposome composition is composed of 20–94 mole percenthydrogenated soy phosphatidylcholine, 1–20 mole percent distearoylphosphatidylcholine derivatized with polyethyleneglycol and 5–60 molepercent cholesterol.

Another preferred composition is 30–65 mole percent hydrogenated soyphosphatidylcholine, 5–20 mole percent distearoyl phosphatidylcholinederivatized with polyethyleneglycol and 30–50 mole percent cholesterol.

In another aspect, the invention includes a composition foradministration of a topoisomerase I inhibitor or a topoisomerase I/IIinhibitor, comprising liposomes composed of vesicle-forming lipids andhaving an inside/outside ion gradient effective to retain the drugwithin the liposomes. Entrapped in the liposomes is the topoisomerase Iinhibitor or the topoisomerase I/II inhibitor at a concentration of atleast about 0.20 μmole drug per μmole lipid.

In another aspect, the invention includes a method of treating a tumorin a subject, comprising preparing liposomes composed of vesicle-forminglipids including between 1–20 mole percent of a vesicle-forming lipidderivatized with a hydrophilic polymer chain, the liposomes being formedunder conditions that distribute the polymer on both sides of theliposomes' bilayer membrane. The liposomes contain a topoisomerase Iinhibitor or a topoisomerase I/II inhibitor entrapped in the liposomesat a concentration of at least about 0.10 mole per μmole lipid, theliposomes having an inside/outside ion gradient sufficient to retain thetopoisomerase I inhibitor or topoisomerase I/II inhibitor within theliposome at the specified concentration. The liposomes are thenadministered to the subject.

In one embodiment of this aspect, the method further includes entrappingthe topoisomerase I inhibitor or topoisomerase I/II inhibitor in theliposomes by remote loading, for example, via an ammonium sulfategradient.

These and other objects and features of the invention will be more fullyappreciated when the following detailed description of the invention isread in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A is a plot of the blood circulation lifetime ofliposome-entrapped MPE-camptothecin (solid circles), taken as thepercent of injected dose as a function of time, compared to the freeform of the drug (solid squares);

FIG. 1B shows the blood concentration of MPE-camptothecin, as a functionof time, in hours, after administration of liposome-entrappedMPE-camptothecin (solid circles) and of free (non-liposomal)MPE-camptothecin (solid squares) to rats;

FIG. 2A is a plot showing the body weight of mice, in grams, as afunction of days after tumor inoculation with an HT29 colon tumor. Theanimals were treated on days 10, 16 and 23 after tumor inoculation withliposome-entrapped MPE-camptothecin at dosages of 24 mg/kg (closedcircles), 15 mg/kg (closed triangles) and 6 mg/kg (closed squares) andwith free MPE-camptothecin at doses of 24 mg/kg (open circles), 15 mg/kg(open triangles) and 6 mg/kg (open squares);

FIG. 2B is a plot showing tumor volume, in mm³, as a function of daysafter inoculation with an HT29 colon tumor. The animals were treated ondays 10, 16 and 23 after tumor inoculation with liposome-entrappedMPE-camptothecin at dosages of 24 mg/kg (closed circles), 15 mg/kg(closed triangles) and 6 mg/kg (closed squares) and with free drug atdoses of 24 mg/kg (open circles), 15 mg/kg (open triangles) and 6 mg/kg(open squares);

FIG. 3A is a plot showing the body weight of mice, in grams, as afunction of days after inoculation with an HT29 colon tumor. The animalswere treated on days 9, 16 and 23 after tumor inoculation withliposome-entrapped MPE-camptothecin at dosages of 5 mg/kg (opentriangles), 3 mg/kg (open inverted triangles), 1 mg/kg (open diamonds),0.5 mg/kg (open circles) and 0.1 mg/kg (open squares) and with freeMPE-camptothecin at a dose of 20 mg/kg (closed squares);

FIG. 3B is a plot showing tumor volume, in mm³, as a function of daysafter inoculation with an HT29 colon tumor. The animals were treated ondays 9, 16 and 23 after tumor inoculation with liposome-entrappedMPE-camptothecin at dosages of 5 mg/kg (open triangles), 3 mg/kg (openinverted triangles), 1 mg/kg (open diamonds), 0.5 mg/kg (open circles)and 0.1 mg/kg (open squares) and with free MPE-camptothecin at a dose of20 mg/kg (closed squares);

FIGS. 4A–4B are plots showing the plasma concentration of topotecan as afunction of time, in hours, after administration of liposome-entrappedtopotecan (solid triangles) and of free (non-liposomal) topotecan (solidsquares) to rats at dosages of 2 mg/kg (FIG. 4A) and 5 mg/kg (FIG. 4B);

FIG. 5A is a plot showing the body weight of mice, in grams, as afunction of days after inoculation with an HT29 colon tumor.

The animals were treated on days 9, 16 and 23 after tumor inoculationwith liposome-entrapped topotecan at dosages of 2 mg/kg (diamonds), 5mg/kg (circles), 8 mg/kg (open squares); liposome-entrappedMPE-camptothecin at 4 mg/kg (triangles); free topotecan at a dose of 25mg/kg (inverted triangles) and saline (closed squares);

FIG. 5B is a plot showing tumor volume, in mm³, as a function of daysafter inoculation with an HT29 colon tumor. The animals were treated ondays 9, 16 and 23 after tumor inoculation with liposome-entrappedtopotecan at dosages of 2 mg/kg (diamonds), 5 mg/kg (circles), 8 mg/kg(open squares); liposome-entrapped MPE-camptothecin at 4 mg/kg(triangles); free topotecan at a dose of 25 mg/kg (inverted triangles)and saline (closed squares);

FIG. 6 is a plot of plasma concentration of CKD602 as a function oftime, in hours, after administration of liposome-entrapped CKD602 (solidcircles) and of free (non-liposomal) topotecan (solid squares) to ratsat a dosage of 1 mg/kg;

FIG. 7A is a plot showing the body weight of mice, in grams, as afunction of days after inoculation with an HT29 colon tumor.

The animals were treated on days 9, 16 and 23 after tumor inoculationwith liposome-entrapped CKD602 at dosages of 4 mg/kg (diamonds), 2 mg/kg(circles), 1 mg/kg (open squares); liposome-entrapped MPE-camptothecinat 4 mg/kg (triangles); free CKD602 at a dose of 20 mg/kg (invertedtriangles) and saline (closed squares); and

FIG. 7B is a plot showing tumor volume, in mm³, as a function of daysafter inoculation with an HT29 colon tumor. The animals were treated ondays 9, 16 and 23 after tumor inoculation with liposome-entrapped CKD602at dosages of 4 mg/kg (diamonds), 2 mg/kg (circles), 1 mg/kg (opensquares); liposome-entrapped MPE-camptothecin at 4 mg/kg (triangles);free CKD602 at a dose of 20 mg/kg (inverted triangles) and saline(closed squares).

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

Unless otherwise indicated, the terms below have the following meaning:

“Effective amount” or “effective dose” refers to the amount necessary orsufficient to inhibit undesirable cell growth, e.g., prevent undesirablecell growth or reduce existing cell growth, such as tumor cell growth.The effective amount can vary depending on factors known to those ofskill in the art, such as the type of cell growth, the mode and regimenof administration, the size of the subject, the severity of the cellgrowth, etc. One of skill in the art would be able to consider suchfactors and make the determination regarding the effective amount.

“Therapeutically effective antitumor therapy” refers to a therapy whichis effective to maintain or decrease the size, e.g., volume, of aprimary tumor or metastatic tumor.

“Topoisomerase I inhibitor” refers to any compound that inhibits orreduces the action of topoisomerase I enzyme.

“Topoisomerase I/II inhibitor” refers to any compound that inhibits orreduces the action of both topoisomerase I enzyme and topoisomerase IIenzyme.

“Topoisomerase inhibitor” refers to a topoisomerase I inhibitor or atopoisomerase I/II inhibitor.

“MPE-camptothecin” refers to7-(4-methyl-piperazino-methylene)-10,11-ethylenedioxy-20(S)-camptothecin.

“Topotecan” refers to 9-dimethyl-aminomethyl-10-hydroxycamptothecin.

“CKD-602” refers to 7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin.

II. Liposome Composition

The present invention is directed to a liposome composition foradministration of a topoisomerase I inhibitor or a topoisomerase I/IIinhibitor. In studies performed in support of the invention, threetopoisomerase inhibitors were entrapped in liposomes and characterizedin vivo: topotecan,7-(4-methyl-piperazino-methylene)-10,11-ethylenedioxy-20(S)-camptothecin(referred to hereing as “MPE-camptothecin”) and7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin (referred to herein as“CKD-602”). The drugs were entrapped in liposomes by remote loading toachieve a high drug load stably retained in the liposomes, as will bedescribed. In vivo studies with the formulations demonstrated that theliposome composition achieves a surprising and unexpected degree ofimprovement in therapeutic activity when compared to therapy with thetopoisomerase inhibitor in free form. More specifically, and as will bedescribed below, the dose of the liposome-entrapped topoisomerase Iinhibitor MPE-camptothecin required to achieve therapeutic antitumortherapy is about 20 times lower than the dose required when the drug isadministered in free form.

In this section, the liposome composition will be described, includingmethods for preparing the liposomes.

A. Liposome Components

Liposomes suitable for use in the composition of the present inventioninclude those composed primarily of vesicle-forming lipids.Vesicle-forming lipids can form spontaneously into bilayer vesicles inwater, as exemplified by the phospholipids. The liposomes can alsoinclude other lipids incorporated into the lipid bilayers, with thehydrophobic moiety in contact with the interior, hydrophobic region ofthe bilayer membrane, and the head group moiety oriented toward theexterior, polar surface of the bilayer membrane.

The vesicle-forming lipids are preferably ones having two hydrocarbonchains, typically acyl chains, and a head group, either polar ornonpolar. There are a variety of synthetic vesicle-forming lipids andnaturally-occurring vesicle-forming lipids, including the phospholipids,such as phosphatidylcholine, phosphatidylethanolamine, phosphatidicacid, phosphatidylinositol, and sphingomyelin, where the two hydrocarbonchains are typically between about 14–22 carbon atoms in length, andhave varying degrees of unsaturation. The above-described lipids andphospholipids whose acyl chains have varying degrees of saturation canbe obtained commercially or prepared according to published methods.Other suitable lipids include glycolipids and sterols such ascholesterol.

Cationic lipids are also suitable for use in the liposomes of theinvention, where the cationic lipid can be included as a minor componentof the lipid composition or as a major or sole component.

Such cationic lipids typically have a lipophilic moiety, such as asterol, an acyl or diacyl chain, and where the lipid has an overall netpositive charge. Preferably, the head group of the lipid carries thepositive charge. Exemplary cationic lipids include1,2-dioleyloxy-3-(trimethylamino) propane (DOTAP);N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammoniumbromide (DMRIE); N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA);3β[N-(N′,N′-dimethylaminoethane) carbamoly]cholesterol (DC-Chol); anddimethyldioctadecylammonium (DDAB).

The cationic vesicle-forming lipid may also be a neutral lipid, such asdioleoylphosphatidyl ethanolamine (DOPE) or an amphipathic lipid, suchas a phospholipid, derivatized with a cationic lipid, such as polylysineor other polyamine lipids. For example, the neutral lipid (DOPE) can bederivatized with polylysine to form a cationic lipid.

In another embodiment, the vesicle-forming lipid is selected to achievea specified degree of fluidity or rigidity, to control the stability ofthe liposome in serum and to control the rate of release of theentrapped agent in the liposome.

Liposomes having a more rigid lipid bilayer, or a liquid crystallinebilayer, are achieved by incorporation of a relatively rigid lipid,e.g., a lipid having a relatively high phase transition temperature,e.g., above room temperature, more preferably above body temperature andup to 80° C. Rigid, i.e., saturated, lipids contribute to greatermembrane rigidity in the lipid bilayer. Other lipid components, such ascholesterol, are also known to contribute to membrane rigidity in lipidbilayer structures.

On the other hand, lipid fluidity is achieved by incorporation of arelatively fluid lipid, typically one having a lipid phase with arelatively low liquid to liquid-crystalline phase transitiontemperature, e.g., at or below room temperature, more preferably, at orbelow body temperature.

Vesicle-forming lipids having a main phase transition temperatures fromapproximately 2° C.–80° C. are suitable for use as the primary liposomecomponent of the present composition. In a preferred embodiment of theinvention, a vesicle-forming lipid having a main phase transitiontemperature above about 37° C. is used as the primary lipid component ofthe liposomes. In another preferred embodiment, a lipid having a phasetransition temperature between about 37–70° C. is used. By way ofexample, the lipid distearoyl phosphatidylcholine (DSPC) has a mainphase transition temperature of 55.1° C. and the lipid hydrogenated soyphosphatidylcholine (HSPC) has a phase transition temperature of 58° C.Phase transition temperatures of many lipids are tabulated in a varietyof sources, such as Avanti Polar Lipids catalogue and Lipid ThermotropicPhase Transition Database (LIPIDAT, NIST Standard Reference Database34).

The liposomes also include a vesicle-forming lipid derivatized with ahydrophilic polymer. As has been described, for example in U.S. Pat. No.5,013,556 and in WO 98/07409, which are hereby incorporated byreference, such a hydrophilic polymer provides a surface coating ofhydrophilic polymer chains on both the inner and outer surfaces of theliposome lipid bilayer membranes. The outermost surface coating ofhydrophilic polymer chains is effective to provide a liposome with along blood circulation lifetime in vivo. The inner coating ofhydrophilic polymer chains extends into the aqueous compartments in theliposomes, i.e., between the lipid bilayers and into the central corecompartment, and is in contact with the entrapped compound after thecompound is loaded via remote loading. As will be illustrated below, theliposome formulation having a surface coating of hydrophilic polymerchains distributed on the inner and outer liposome surfaces provides fora topoisomerase I inhibitor or topoisomerase I/II inhibitor compositionwhere the compound is retained in the liposomes for improved therapeuticactivity.

Vesicle-forming lipids suitable for derivatization with a hydrophilicpolymer include any of those lipids listed above, and, in particularphospholipids, such as distearoyl phosphatidylethanolamine (DSPE).

Hydrophilic polymers suitable for derivatization with a vesicle-forminglipid include polyvinylpyrrolidone, polyvinylmethylether,polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline,polyhydroxypropylmethacrylamide, polymethacrylamide,polydimethylacrylamide, polyhydroxypropylmethacrylate,polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose,polyethyleneglycol, and polyaspartamide. The polymers may be employed ashomopolymers or as block or random copolymers.

A preferred hydrophilic polymer chain is polyethyleneglycol (PEG),preferably as a PEG chain having a molecular weight between 500–10,000daltons, more preferably between 500–5,000 daltons, most preferablybetween 1,000–2,000 daltons. Methoxy or ethoxy-capped analogues of PEGare also preferred hydrophilic polymers, commercially available in avariety of polymer sizes, e.g., 120–20,000 daltons.

Preparation of vesicle-forming lipids derivatized with hydrophilicpolymers has been described, for example in U.S. Pat. No. 5,395,619.Preparation of liposomes including such derivatized lipids has also beendescribed, where typically, between 1–20 mole percent of such aderivatized lipid is included in the liposome formulation. It will beappreciated that the hydrophilic polymer may be stably coupled to thelipid, or coupled through an unstable linkage which allows the coatedliposomes to shed the coating of polymer chains as they circulate in thebloodstream or in response to a stimulus.

B. Topoisomerase Inhibitor

The liposomes of the invention include a topoisomerase inhibitorentrapped in the liposome. Entrapped is intended to includeencapsulation of an agent in the aqueous core and aqueous spaces ofliposomes. It will be appreciated that for compounds having somehydrophobicity, entrapment in the lipid bilayer(s) of the liposomes mayalso occur.

Topoisomerases catalyze the introduction and relaxation of superhelicityin DNA. Several types of enzymes with varying specifities are known tobe important in the replication of DNA, as well as in the repair,genetic recombination and transcription of DNA. The simplesttopoisomerases, designated topoisomerase I, relax superhelical DNA, aprocess that is energetically spontaneous. The gyrases, which are knownas topoisomerase II, catalyze the energy-requiring and ATP-dependentintroduction of negative superhelical twists into DNA. In DNAreplication, topoisomerases I and II have the function of relaxing thepositive superhelicity that is introduced ahead of the replicating forksby the action of helicases. In addition, gyrases introduce negativetwists into segments of DNA that allow single-strand regions to appear.

Topoisomerase inhibitors, then, are compounds that inhibit topoiosmeraseactivity. Compounds known as topoisomerase I inhibitors have activityagainst topoisomerase I, and the topoiosmerase II inhibitors haveactivity against topoisomerase II. Some compounds have activity againstboth topoisomerase I and topoisomerase II and are known as topoisomeraseI/II inhibitors.

Preferred topoisomerase I inhibitors for use in the present inventionare camptothecin and analogs of camptothecin. Camptothecin is apentacyclic alkaloid initially isolated from the wood and bark ofCamptotheca acuminata, a tree indigenous to China (Wall, M. E. et al.,J. Am. Chem. Soc., 94:388 (1966)). Camptothecin exerts itspharmacological effects by irreversibly inhibiting topoisomerase I.Methods for the synthesis of camptothecin and camptothecin analogs orderivatives are known, and are summarized and set forth in U.S. Pat. No.5,244,903, which is herein incorporated by reference in its entirety.

Analogues of camptothecin include SN-38((+)-(4S)-4,11-diethyl-4,9-dihydroxy-1H-pyrano[3′,4′:6,7]-indolizino[1,2-b]quinoline-3,14(4H,12H)-dione);9-aminocamptothecin; topotecan (hycamtin;9-dimethyl-aminomethyl-10-hydroxycamptothecin); irinotecan (CPT-11;7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxy-camptothecin),which is hydrolyzed in vivo to SN-38); 7-ethylcamptothecin and itsderivatives (Sawada, S. et al., Chem. Pharm. Bull., 41(2):310–313(1993)); 7-chloromethyl-10,11-methylene-dioxy-camptothecin; and others(SN-22, Kunimoto, T. et al., J. Pharmacobiodyn., 10(3):148–151 (1987);N-formylamino-12,13,dihydro-1,11-dihydroxy-13-(beta-D-glucopyranosyl)-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione(NB-506, Kanzawa, F et al., Cancer Res., 55(13):2806–2813 (1995);DX-8951f and lurtotecan (GG-211 or7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20(S)-camptothecin)(Rothenberg, M. L., Ann. Oncol., 8(9):837–855 (1997)) and7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin (CKD602, Chong Kun DangCorporation, Seoul Korea).

Topoisomerase inhibitors having activity against both topoisomerase Iand topoisomerase II include6-[[2-(dimethylamino)-ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-onedihydrochloride, (TAS-103, Utsugi, T., et al., Jpn. J. Cancer Res.,88(10):992–1002 (1997)) and3-methoxy-11H-pyrido[3′,4′–4,5]pyrrolo[3,2-c]quinoline-1,4-dione(AzalQD, Riou, J. F., et al., Mol. Pharmacol., 40(5):699–706 (1991)).

In one embodiment of the invention, the topoisomerase I inhibitoradministered is the pharmacologically active enantiomer of acamptothecin analogue having a chiral center. The enantiomer can beresolved from the racemic mixture using techniques known to those ofskill in the art.

C. Method of Preparing the Liposome Composition

The liposomes may be prepared by a variety of techniques, such as thosedetailed in Szoka, F., Jr., et al., Ann. Rev. Biophys. Bioeng. 9:467(1980), and specific examples of liposomes prepared in support of thepresent invention will be described below. Typically, the liposomes aremultilamellar vesicles (MLVs), which can be formed by simple lipid-filmhydration techniques. In this procedure, a mixture of liposome-forminglipids and including a vesicle-forming lipid derivatized with ahydrophilic polymer are dissolved in a suitable organic solvent which isevaporated in a vessel to form a dried thin film. The film is thencovered by an aqueous medium to form MLVs, typically with sizes betweenabout 0.1 to 10 microns. Exemplary methods of preparing derivatizedlipids and of forming polymer-coated liposomes have been described inco-owned U.S. Pat. Nos. 5,013,556, 5,631,018 and 5,395,619, which areincorporated herein by reference.

The therapeutic agent of choice can be incorporated into liposomes bystandard methods, including (i) passive entrapment of a water-solublecompound by hydrating a lipid film with an aqueous solution of theagent, (ii) passive entrapment of a lipophilic compound by hydrating alipid film containing the agent, and (iii) loading an ionizable drugagainst an inside/outside liposome ion gradient, termed remote loading.Other methods, such as reverse evaporation phase liposome preparation,are also suitable.

In the present invention, a preferred method of preparing the liposomesis by remote loading. In the studies performed in support of theinvention, three exemplary topoisomerase I inhibitors were loaded intopre-formed liposomes by remote loading against an ion concentrationgradient, as has been described in the art (U.S. Pat. No. 5,192,549) andas described in Example 1. In a remote loading procedure, a drug isaccumulated in the liposomes' central compartment at concentrationlevels much greater than can be achieved with other loading methods. Ina preferred embodiment of the invention, the topoisomerase I inhibitoror topoisomerase I/II inhibitor is loaded into the liposomes to aconcentration of at least about 0.10 μmole drug per μmole lipid, morepreferably of at least about 0.15 μmole drug per μmole lipid, mostpreferably of at least about 0.20 μmole drug per μmole lipid. Theliposomes prepared in support of the invention containedMPE-camptothecin, topotecan or CKD602. As set forth in Example 1, thesecompounds were loaded into the liposomes by remote loading, discussedbelow, to a drug concentration level of greater than 0.20 mmole drug perμmole lipid (see the table in Example 1).

Liposomes having an ion gradient across the liposome bilayer for use inremote loading can be prepared by a variety of techniques. A typicalprocedure is as described above, where a mixture of liposome-forminglipids is dissolved in a suitable organic solvent and evaporated in avessel to form a thin film. The film is then covered with an aqueousmedium containing the solute species that will form the aqueous phase inthe liposome interior spaces.

After liposome formation, the vesicles may be sized to achieve a sizedistribution of liposomes within a selected range, according to knownmethods. The liposomes are preferably uniformly sized to a selected sizerange between 0.04 to 0.25 μm.

Small unilamellar vesicles (SUVs), typically in the 0.04 to 0.08 μmrange, can be prepared by extensive sonication or homogenization of theliposomes. Homogeneously sized liposomes having sizes in a selectedrange between about 0.08 to 0.4 microns can be produced, e.g., byextrusion through polycarbonate membranes or other defined pore sizemembranes having selected uniform pore sizes ranging from 0.03 to 0.5microns, typically, 0.05, 0.08, 0.1, or 0.2 microns. The pore size ofthe membrane corresponds roughly to the largest size of liposomesproduced by extrusion through that membrane, particularly where thepreparation is extruded two or more times through the same membrane. Thesizing is preferably carried out in the original lipid-hydrating buffer,so that the liposome interior spaces retain this medium throughout theinitial liposome processing steps.

After sizing, the external medium of the liposomes is treated to producean ion gradient across the liposome membrane, which is typically a lowerinside/higher outside concentration gradient. This may be done in avariety of ways, e.g., by (i) diluting the external medium, (ii)dialysis against the desired final medium, (iii) molecular-sievechromatography, e.g., using Sephadex G-50, against the desired medium,or (iv) high-speed centrifugation and resuspension of pelleted liposomesin the desired final medium. The external medium which is selected willdepend on the mechanism of gradient formation and the external pHdesired, as will now be considered.

In the simplest approach for generating an ion gradient, the hydrated,sized liposomes have a selected internal-medium pH. The suspension ofthe liposomes is titrated until a desired final pH is reached, ortreated as above to exchange the external phase buffer with one havingthe desired external pH. For example, the original medium may have a pHof 5.5, in a selected buffer, e.g., glutamate or phosphate buffer, andthe final external medium may have a pH of 8.5 in the same or differentbuffer. The internal and external media are preferably selected tocontain about the same osmolarity, e.g., by suitable adjustment of theconcentration of buffer, salt, or low molecular weight solute, such assucrose.

In another general approach, the gradient is produced by including inthe liposomes, a selected ionophore. To illustrate, liposomes preparedto contain valinomycin in the liposome bilayer are prepared in apotassium buffer, sized, then exchanged with a sodium buffer, creating apotassium inside/sodium outside gradient. Movement of potassium ions inan inside-to-outside direction in turn generates a lower inside/higheroutside pH gradient, presumably due to movement of protons into theliposomes in response to the net electronegative charge across theliposome membranes (Deamer, et al., 1972).

In another more preferred approach, the proton gradient used for drugloading is produced by creating an ammonium ion gradient across theliposome membrane, as described, for example, in U.S. Pat. No.5,192,549. Here the liposomes are prepared in an aqueous buffercontaining an ammonium salt, typically 0.1 to 0.3 M ammonium salt, suchas ammonium sulfate, at a suitable pH, e.g., 5.5 to 7.5. The gradientcan also be produced by using sulfated polymers, such as dextranammonium sulfate or heparin sulfate. After liposome formation andsizing, the external medium is exchanged for one lacking ammonium ions,e.g., the same buffer but one in which ammonium sulfate is replaced byNaCl or a sugar that gives the same osmolarity inside and outside of theliposomes.

After liposome formation, the ammonium ions inside the liposomes are inequilibrium with ammonia and protons. Ammonia is able to penetrate theliposome bilayer and escape from the liposome interior. Escape ofammonia continuously shifts the equilibrium within the liposome towardthe right, to production of protons.

The topoisomerase inhibitor is loaded into the liposomes by adding thedrug to a suspension of the ion gradient liposomes, and the suspensionis treated under conditions effective to allow passage of the compoundfrom the external medium into the liposomes. Incubation conditionssuitable for drug loading are those which (i) allow diffusion of thederivatized compound, with such in an uncharged form, into theliposomes, and (ii) preferably lead to high drug loading concentration,e.g., 5–500 mM drug encapsulated, more preferably between 20–200 mM,most preferably between 50–300 mM.

The loading is preferably carried out at a temperature above the phasetransition temperature of the liposome lipids. Thus, for liposomesformed predominantly of saturated phospholipids, the loading temperaturemay be as high as 60° C. or more. The loading period is typicallybetween 15–120 minutes, depending on permeability of the drug to theliposome bilayer membrane, temperature, and the relative concentrationsof liposome lipid and drug.

With proper selection of liposome concentration, external concentrationof added compound, and the ion gradient, essentially all of the compoundmay be loaded into the liposomes.

For example, with a pH gradient of 3 units (or the potential of such agradient employing an ammonium ion gradient), the finalinternal:external concentration of drug will be about 1000:1. Knowingthe calculated internal liposome volume, and the maximum concentrationof loaded drug, one can then select an amount of drug in the externalmedium which leads to substantially complete loading into the liposomes.

Alternatively, if drug loading is not effective to substantially depletethe external medium of free drug, the liposome suspension may betreated, following drug loading, to remove non-encapsulated drug. Freedrug can be removed, for example, by molecular sieve chromatography,dialysis, or centrifugation.

In another embodiment of the invention, the topoisomerase inhibitor isloaded into preformed liposomes which include in the liposome interior atrapping agent effective to complex with the topoisomerase inhibitor andenhance retention of the compound. In a preferred embodiment, thetrapping agent is a polyanionic polymer, e.g., a molecule consisting ofrepetitive units of preferably similar chemical structure and havingionizable groups, that is, chemical functional groups capable ofelectrolytic dissociation resulting in the formation of ionic charge,and preferably an anionic charge. Polymers having a molecular weightover a broad range are suitable, from 400–2,000,000 Daltons.

The polyanionic polymer is entrapped in the liposomes during lipidvesicle formation. Upon loading of a drug into the pre-formed liposomes,the polymer serves to trap or retain the drug within the liposomes. Inthe studies described herein, dextran sulfate was used as an exemplarypolyanionic polymer. Dextran sulfate is a polymer of anhydroglucose withapproximately 2.3 sulfate groups per glucosoyl residue. It is composedof approximately 95% alpha-D-(1–6) linkages and the remaining (1–3)linkages account for the branching of dextran. The polymer is readilyavailable in molecular weights ranging from 5,000 to 500,000 Daltons.However, other polymers are suitable including sulfated, sulfonated,carboxylated or phosphated hydrophilic polymers. For example, sulfatedproteoglycans, such as sulfated heparin, sulfated polysaccharids, suchas sulfated cellulose or cellulose derivatives, carrageenin, mucin,sulfated polypeptides, such as polylysine with sulfated amine groups,glycopeptides with sulfonate-derivatized saccharide or peptide subunits,and hyaluronic acid. Chondroitin sulfates A, B and C, keratin sulfates,dermatan sulfates are also contemplated. The polymer can also be aneutral polymer modified to include an anionic functional group. Forexample, amylose, pectin, amylopectin, celluloses, and dextran can bemodified to include an anionic subunit. Polymers bearing a sulfo groupsuch as polyvinylsulfate, polyvinylsulfonate polystyrenesulfonate andsulfated rosin gum are also suitable.

Preparation of liposomes which include such a trapping agent isdescribed with respect to Example 4. In this example, the polyanionicpolymer dextran sulfate is entrapped in the liposomes by adding theliposome lipids, which are first dissolved in ethanol, to a solution ofdextran sulfate ammonium salt and mixed to form liposomes having dextransulfate ammonium salt entrapped within the liposomes. The external mediawas exchanged to establish an ammonium ion gradient across the liposomesfor remote loading of drug.

III. In Vivo Administration of the Composition

Liposomes were prepared in support of the invention as described inExample 1. The topoisomerase I inhibitors(7-(4-methylpiperazino)-methylene)-10,11-ethylenedioxy-20(S)-camptothecin),referred to herein as “MPE-camptothecin”; topotecan; and7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin, referred to herein as“CKD-602”, were loaded into liposomes under an ammonium sulfate ionconcentration gradient. The liposomes were composed of hydrogenated soyphosphatidylcholine, cholesterol and polyethylene glycol derivatized todistearoyl phosphatidylethanolamine (PEG-DSPE) in a molar ratio55.4:39:5.6.

The table in Example 1 summarizes the drug to lipid ratios for theliposome formulations prepared. The calculated liposomal drugconcentrations for the three compounds, based on an extruded liposomecaptured volume of 0.9 μl/μmole lipid, are 284 mM for MPE-camptothecin,264 mM for topotecan and 298 for CKD-602. Based on an extruded liposomecaptured volume of 1.5 μl/μmole lipid, the calculated liposomal drugconcentrations are 189 mM for MPE-camptothecin, 174 mM for topotecan and198 for CKD-602. The in vivo studies performed with each drug will nowbe described.

A. In Vivo Administration of MPE-Camptothecin

The long-circulating, PEG-coated liposomes containing MPE-camptothecinwere administered to rats to determine the blood circulation lifetime ofthe drugs in liposome-entrapped form.

The pharmacokinetic profile of the liposome-entrapped drug and of thefree drug are shown in FIG. 1A as the percent of injected dose as afunction of time. As can be seen, the blood circulation time of thetopoisomerase I inhibitor in liposome-entrapped form (solid circles) issignificantly longer than the free form of the drug (solid squares). ForMPE-camptothecin, the blood circulation half-life of theliposome-entrapped drug was 14 hours, compared to about 50 minutes forthe free drug. The blood clearance of the liposome-entrapped drug inrats was approximately 35-fold lower and the area under the curve wasapproximately 1250-fold higher than that of the free drug. Analyticalresults indicate that essentially all the drug remains entrapped in theliposomes in the bloodstream.

FIG. 1B shows the concentration of MPE-camptothecin in whole blood afteradministration of the liposome formulation (solid circles) and of thefree drug to rats. The longer circulation lifetime results in a higherconcentration of the drug in the blood.

The anti-tumor efficacy of the MPE-camptothecin liposome formulation wasdetermined in xenograft tumor models, where homozygous nude mice wereinoculated with human tumor cells of colon, HT29 origin. Surprisingly,these toxicity and antitumor efficacy studies showed that liposomalMPE-camptothecin was significantly more toxic than the free form of thedrug at equivalent doses. These studies and the results will now bedescribed.

Liposomes were prepared as set forth in Example 1 to include entrappedMPE-camptothecin. Nude mice with HT-29 colon xenografts were treatedwith liposome-entrapped MPE-camptothecin at dosages of 24 mg/kg, 15mg/kg and 6 mg/kg or with free MPE-camptothecin at the same dosages.Treatment began 10 days after tumor inoculation and doses wereadministered at days 10, 16 and 23. The tumor volume in each animal wasassessed during and following treatment as described in Example 2.

The body weight of each test animal and the tumor volume of each animalare shown, respectively in FIGS. 2A and 2B, where animals were treatedwith liposomal entrapped MPE-camptothecin at dosages of 24 mg/kg (closedcircles), 15 mg/kg (closed triangles) and 6 mg/kg (closed squares) andwith free MPE-camptothecin at doses of 24 mg/kg (open circles), 15 mg/kg(open triangles) and 6 mg/kg (open squares).

With respect to the animals treated with the liposome-entrappedMPE-camptothecin, all of the animals dosed with 15 mg/kg and 24 mg/kgdied after two doses due to drug-related toxicity, with most deaths onday five after the first dose. All of the animals treated with 6 mg/kgliposome-entrapped MPE-camptothecin survived until administration of thethird dose on day 23, after which five of the ten animals died within afew days. The toxicity of the liposome-entrapped MPE-camptothecin isreflected in the greater body weight losses, as seen in FIG. 2A.

In contrast, all of the animals treated with the free form of the drugsurvived the study, with the exception of one animal in the 24 mg/kgdosing group that died a few days after the third dose on day 23.

TABLE 1 Number of Surviving Animals Number of after after after DoseTest dose 1 dose 2 dose 3 Treatment mg/kg Animals (day 9) (day 16) (day23) Saline na 20 20 20 20 free MPE- 24 10 10 10 9 camptothecin free MPE-15 10 10 10 10 camptothecin free MPE- 6 10 10 10 10 camptothecinliposome-entrapped 24 10 1 0 0 liposome-entrapped 15 10 5 0 0liposome-entrapped 6 10 10 10 5

With respect to antitumor activity of the formulations, theliposome-entrapped MPE-camptothecin was more effective than the freeform of the drug in inhibiting tumor growth, despite its greatertoxicity. This can be seen in FIG. 2B, where the 6 mg/kg dose ofliposome-entrapped MPE-camptothecin was significantly more effective ininhibiting tumor growth (log growth rate of −0.026) than even thehighest dose level of free MPE-camptothecin (24 mg/kg, log growth rate0.0048).

The complete and partial remission of the tumors in the test animals wasmonitored and is presented in Table 2. Complete remission of a tumor isdefined as the elimination of tumor mass until the end of theexperiment. A partial remission is defined as a tumor volume of lessthan 50% of the peak tumor volume for an individual animal.

TABLE 2 Dose Partial Treatment mg/kg Complete Remission¹ Remission²Saline  0/20 0/20 free MPE-camptothecin 24  3/10 1/10 freeMPE-camptothecin 15  2/10 0/10 free MPE-camptothecin 6  0/10 0/10liposome-entrapped 24 —³ —³ liposome-entrapped 15 —³ —³liposome-entrapped 6 10/10 na⁴ ¹complete remission defined aselimination of tumor mass until experiment termination. ²partialremission defined as a tumor volume of less than 50% of the peak tumorvolume for an individual animal. ³all 10 animals in test groups diedafter the second dose on day 16. ⁴na = not applicable

As can be seen in Table 2, the liposome-entrapped MPE-camptothecin at adose of 6 mg/kg was effective to cause a complete remission of tumors inall 10 test animals. This effect was observed within five days after thesecond treatment on day 16. As noted above, five of the test animals inthe 6 mg/kg liposome-entrapped test group died shortly after the thirddose.

In the surviving five animals, the tumors did not recur by the end ofthe study, approximately 30 days after the final treatment on day 23.Data is unavailable for the animals treated with 15 mg/kg and 24 mg/kgliposome-entrapped MPE-camptothecin, since all of the animals in thesetest groups died due to drug-related toxicity, as noted above.

Administration of MPE-camptothecin in free form at a dose of 24 mg/kgresulted in 3 animals with complete tumor remission and 1 animal withpartial tumor remission, as seen in Table 2.

Comparison of the results observed for the drug administered in freeform and in liposome-entrapped form indicate that the drug is morepotent when administered in liposome-entrapped form.

In fact, the liposome-entrapped drug is at least four times more potentthan the free form of the drug, as can be seen by comparing the resultsobtained for a 6 mg/kg of liposome-entrapped MPE-camptothecin dosage toa 24 mg/kg free MPE-camptothecin dosage (FIG. 2B, Table 2). It is clearfrom these results that the dose of liposome-entrapped MPE-camptothecinrequired for therapeutically effective anti-tumor therapy is four timeslower than the dose required when the drug is administered in free form.

Example 2 describes the details of a second study to determine themaximum tolerated dose and the lowest effective dose of theliposome-entrapped MPE-camptothecin. In this study, liposomes wereprepared as described in Example 1 and the liposome formulation wasadministered to test animals at drug dosages of 0.1 mg/kg, 0.5 mg/kg, 1mg/kg, 3 mg/kg and 5 mg/kg. The free drug was administered at 20 mg/kgas a comparison.

Table 3 summarizes the number of test animals in each group, specifyingthe number of animals surviving at each dosing phase of the study. Asseen in the table, all of the control, saline treated animals and all ofthe animals treated with free MPE-camptothecin survived for the durationof the study. Of the ten animals treated with 5 mg/kg liposome-entrappedMPE-camptothecin, four of the animals died of drug-related toxicity andone additional animal died of apparently nonspecific causes after thethird dose. One of the ten animals in the test group receiving 3 mg/kgliposome-entrapped MPE-camptothecin died after the second dose, but thedeath was not considered due to drug treatment because of the absence ofany correlating signs of toxicity. All other animals treated withliposome-entrapped MPE-camptothecin survived the entire study duration.

TABLE 3 Number of Surviving Animals Number of after after after DoseTest dose 1 dose 2 dose 3 Treatment mg/kg Animals (day 9) (day 16) (day23) Saline 20 20 20 20 free MPE-campto- 20 10 10 10 10 thecinliposome-entrapped 5 10 10 10 5 liposome-entrapped 3 10 10 9 9liposome-entrapped 1 10 10 10 10 liposome-entrapped 0.5 10 10 10 10liposome-entrapped 0.1 10 10 10 10

The results of the study are shown in FIGS. 3A–3B, where FIG. 3A showsthe body weight of mice, in grams, as a function of days afterinoculation with the HT-29 colon tumor. The animals were treated on days9, 16 and 23 after tumor inoculation with liposomal entrappedtopoisomerase I inhibitor at dosages of 5 mg/kg (open triangles), 3mg/kg (open inverted triangles), 1 mg/kg (open diamonds), 0.5 mg/kg(open circles) and 0.1 mg/kg (open squares) and with free drug at a doseof 20 mg/kg (closed squares). As can be seen in FIG. 3A, body weightchanges were dose-related and, these changes were correlated with otherobservations of toxicity.

FIG. 3B is a similar plot showing tumor volume, in mm³, as a function ofdays after tumor inoculation, where the dosages are represented by thesame symbols as in FIG. 3A. FIG. 3B shows that both the 5 mg/kg and 3mg/kg dose levels of liposome-entrapped MPE-camptothecin were moretherapeutically effective in inhibiting tumor growth than the 20 mg/kgdose of the free drug.

Treatment with 20 mg/kg of free MPE-camptothecin (log growth rate of0.011) was approximately equivalent in antitumor activity to the 1 mg/kgdosage level of the drug in liposome-entrapped form (log growth rate of0.017).

Table 4 summarizes the complete and partial tumor remission in the testanimals.

TABLE 4 Complete Partial Dose Remis- Remis- Treatment mg/kg sion¹ sion²Saline 0/20 0/20 free MPE-camptothecin 20 0/10 1/10 liposome-entrappedMPE-camptothecin 5 10/10  na³ liposome-entrapped MPE-camptothecin 3 7/101/10 liposome-entrapped MPE-camptothecin 1 0/10 0/10 liposome-entrappedMPE-camptothecin 0.5 0/10 1/10 liposome-entrapped MPE-camptothecin 0.10/10 0/10 ¹Complete remission defined as elimination of tumor mass untilexperiment termination. ²Partial remission defined as a tumor volume ofless than 50% of the peak tumor volume for an individual animal. ³na =not applicable

There were no complete tumor remissions in the animals treated with 20mg/kg of free MPE-camptothecin. In contrast, all ten of the animalstreated with liposome-entrapped MPE-camptothecin at the 5 mg/kg dosagelevel had complete remissions. At the 3 mg/kg dosage, seven of theanimals had complete remission of their tumor.

The results from the study of Example 3 shows that antitumor activity ofthe liposome-entrapped topoisomerase inhibitor MPE-camptothecin issignificantly better when compared to the free form of the drug,indicating that the liposome-entrapped form was about 20-fold morepotent since the antitumor activity of the free drug at a dose of 20mg/kg was most comparable to the activity of a 1 mg/kg dose of theliposome-entrapped form of the drug. That the 3 mg/kg and 5 mg/kgliposome-entrapped MPE-camptothecin dosages were significantly moreeffective in antitumor therapy than the 20 mg/kg dose of the drug infree form indicates that the therapeutic index of the drug entrapped inliposomes is approximately four-fold to five-fold higher than the drugin free form.

B. In Vivo Adminstration of Topotecan

In another study performed in support of the invention, topotecan wasentrapped in liposomes composed of DSPC and mPEG-DSPE in a 95:5 molarratio, as described in Example 4. Early studies, not reported here,indicated that topotecan was not readily retained in the liposomes. Thelipid bilayer was selected to use a single component phospholipid havingan acyl chain length close to DSPE in the mPEG-DSPE component. Such abilayer has minimal packing defects which arise from imperfections innearest neighbor interactions in a solid phase bilayer, which havereduced lateral and rotational mobility relative to fluid bilayers. Inaddition, a dextran-sulfate loading battery was used in order to achieveprecipitation of the topotecan in the liposome interior. Other polymers,in particular polyanionic polymers, are suitable for this purpose, suchas chondroitin sulfate A, polyvinylsulfuric acid, and polyphosphoricacid.

The pre-formed liposomes containing dextran ammonium sulfate in thecentral compartment were loaded with topotecan as described in Example4. After loading, unentrapped drug was removed by diafiltration and theliposomes were characterized. The liposomes were loaded to a drug:lipidratio of 0.238 and the liposomes had an average particle diameter of 87nm.

The liposomes containing topotecan were administered intraveneously torats to determine the blood circulation lifetime. FIGS. 4A–4B show theplasma concentration of topotecan as a function of time afteradministration to rats. FIG. 4A compares the concentration ofliposome-entrapped topotecan administered at 2 mg/kg (solid triangles)to the concentration of free topotecan administered at the same dosage(solid squares). FIG. 4B compares the two forms of the drug at a dosageof 5 mg/kg. The calculated pharmacokinetic parameters are given in Table5.

TABLE 5 Dosage = 2 mg/kg Dosage = 5 mg/kg Free Liposome- Free Liposome-Parameter Topotecan Entrapped Topotecan Entrapped Plasma Cmax (μg/mL)2.89 54.5 8.23 119.3 AUC (μg/mL h) 0.57 523 1.57 1140 T ½ (h) 0.20 7.20.30 9.8 CL (mL/h) 887 0.96 820 1.10 Vol. Dist. (mL) 173 9.2 278 17.5elimination rate 3.45 0.096 2.33 0.071 constant (1/h)

The data in Table 5 shows that the liposome-entrapped drug has asignificantly longer circulation time than the free form of the drug.

The efficacy of the liposomes was determined in another study. Asdescribed in Example 4, the liposomes were administered to mice bearinga subcutaneous xenograft tumor. Tumor-bearing mice were randomized intosix treatment groups of 12 mice for treatment with one of the following:saline, liposome-entrapped MPE-camptothecin 4 mg/kg; free topotecan 25mg/kg; liposome entrapped topotecan at drug dosages of 2 mg/kg, 5 mg/kgor 8 mg/kg. All treatments were administered as intravenous bolusinjections given weekly for 3 treatments, specifically on days 9, 16 and23.

The tumor size in each animal was measured twice weekly during the studyto evaluate therapeutic efficacy. Body weight of each animal wasmonitored twice weekly to assess toxicity of the formulations. Theresults are shown in Tables 6 and 7 and in FIGS. 5A–5B.

TABLE 6 Complete Partial Non- Dose Remis- Remis- Respon- Treatment mg/kgsion¹ sion² sive³ Saline 0 0 12 liposome-entrapped MPE- 4 8 4 0camptothecin free topotecan 25 0 1 11 liposome-entrapped topotecan 2 1 29 liposome-entrapped topotecan 5 2 8 2 liposome-entrapped topotecan 8 73 2 ¹Complete remission defined as elimination of tumor mass untilexperiment termination. ²Partial remission defined as a tumor volume ofless than 50% of the peak tumor volume for an individual animal.³Non-responsive defined as a tumor volume equal to or greater thaninitial tumor volume.

As can be seen from FIGS. 5A and Table 6, left untreated the tumors grewat a rate of 17.8 mm³ per day for the duration of the study. The animalstreated with liposome-entrapped MPE-camptothecin (positive controlanimals) experienced a tumor growth rate −1.2 mm³ per day for theduration of the study. Animals treated with nonencapsulated topotecan,which was administered at 25 mg/kg somewhat below the maximum tolerateddosage (MTD) of 40 mg/kg, had tumor growth of 14.1 mm³ per day. Animalstreated with liposome-entrapped topotecan had tumor growth of 0.9 mm³per day for a dosage of 2 mg/kg, −1.9 mm³ per day for a dosage of 5mg/kg and −0.8 mm³ per day for a dosage of 8 mg/kg. The negative growthrate indicates regression of tumor size below the starting tumor volume.

The size of treated tumors as a function of the size of control tumors(% T/C) was examined for all treatment groups and is summarized in Table6. The National Cancer Institute defines significant anti-tumor activityas a % T/C less than 42.

TABLE 7 Dose % T/C¹ % T/C % T/C Treatment mg/kg Day 29 Day 33 Day 36liposome-entrapped MPE- 4 1.8 0.6 1.9 camptothecin free topotecan 2582.8 79.0 85.9 liposome-entrapped topotecan 2 19.5 12.9 16.3liposome-entrapped topotecan 5 10.5 5.6 5.6 liposome-entrapped topotecan8 2.0 2.2 2.2 ¹% T/C defined as the average tumor volume at dayindicated over the average tumor volume of the control, saline treatedanimals.

C. In Vivo Adminstration of CKD-602

Example 5 describes another study conducted in support of the inventionusing the topoisomerase inhibitor CKD-602. The drug was remotely loadedinto liposomes against an ammonium-sulfate gradient with dextran as atrapping agent. The liposome lipid composition was identical to thatused for the study using topotecan—HSPC and mPEG-DSPE in a 95/5 moleratio.

FIG. 6 is a plot showing the plasma concentration of CKD-602 as afunction of time after administration to rats at a dosage of 1 mg/kg.The liposome-entrapped form of the drug (solid circles) had a calculatedhalf-life of 9.8 hours and an AUC of 274 μg/mL/hr. The free form of thedrug had a calculated half-life of 0.2 hours and an AUC of 0.37μg/mL/hr.

Therapeutic efficacy of the CKD-602 formulation was evaulated using micebearing a HT-29 colon cancer xenograft. Seventy-two mice were inoculatedwith HT-29 tumor cells and nine days later were randomized into sixtreatment groups. The animals in each group were treated with one of thefollowing formulations: saline, liposome-entrapped MPE-camptothecin 4mg/kg; free CKD-602 20 mg/kg; liposome entrapped CKD-602 at drug dosagesof 1 mg/kg, 2 mg/kg or 4 mg/kg. All treatments were administered asintravenous bolus injections given weekly for 3 treatments, specificallyon days 11, 18 and 25.

The tumor size in each animal was measured twice weekly during the studyto evaluate therapeutic efficacy. Body weight of each animal wasmonitored twice weekly to assess toxicity of the formulations. Theresults are shown in Tables 8 and 9 and in FIGS. 7A–7B.

TABLE 8 Complete Partial Non- Dose Remis- Remis- Respon- Treatment mg/kgsion¹ sion² sive³ Saline 0/10 0/10 10/10  liposome-entrapped MPE- 4 6/100/10 4/10 camptothecin free CKD602 20 0/6  0/6  6/6  liposome-entrappedCKD602 1 2/10 7/10 1/10 liposome-entrapped CKD602 2 6/10 2/10 2/10liposome-entrapped CKD602 4 4/4  0/4  0/4  ¹Complete remission definedas elimination of tumor mass until experiment termination. ²Partialremission defined as a tumor volume of less than 50% of the peak tumorvolume for an individual animal. ³Non-responsive defined as a tumorvolume equal to or greater than initial tumor volume.

As can be seen in Table 8 and in FIG. 7B, the animals treated withsaline experienced continuous tumor growth, at a rate of 15.45 mm³ perday for the duration of the study. The animals treated with theliposome-entrapped MPE-camptothecin (positive control animals) had atumor growth rate of −0.63 mm³ per day for the duration of the study.Animals treated with free, unentrapped CKD602 had tumor growth of 15.21mm³ per day. Animals treated with liposomal CKD602 had tumor growth of−2.21 mm³ per day for animals treated with a dose of 1 mg/kg, −0.96 mm³per day for a dose of 2 mg/kg and −2.37 mm³ per day for a dose of 4mg/kg. The negative growth rate indicates regression of tumor size belowthe starting tumor volume.

The size of treated tumors as a function of the size of control tumors(% T/C) was examined for all treatment groups and is summarized in Table9. The National Cancer Institute defines significant anti-tumor activityas a % T/C less than 42.

TABLE 9 Dose % T/C¹ % T/C % T/C Treatment mg/kg Day 29 Day 33 Day 36liposome-entrapped MPE- 4 2.9 2.3 1.6 camptothecin free CKD602 20 129.1120.1 99.9 liposome-entrapped CKD602 1 11.4 7.7 4.4 liposome-entrappedCKD602 2 4.8 2.8 1.6 liposome-entrapped CKD602 4 1.0 1.3 0.9 ¹% T/Cdefined as the average tumor volume at day indicated over the averagetumor volume of the control, saline treated animals.

IV. EXAMPLES

The following examples illustrate methods of preparing, characterizing,and using the composition of the present invention. The examples are inno way intended to limit the scope of the invention.

Materials

The topoisomerase inhibitor(7-(4-methyl-piperazino-methylene)-10,11-ethylenedioxy-20(S)-camptothecintrifluoroacetate (GI147211) (MPE-camptothecin), was provided by GlaxoResearch Institute, Research Triangle Park, N.C. CKD602(7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin) was provided by ChongKun Dang Corporation, Seoul Korea. Topotecan (Hycamtin®) was purchasedcommercially.

Materials for preparation of the liposomes and all other reagents werefrom commercially available sources.

Methods

Animal Studies: Homozygous nude mice were obtained from Taconic Farms(Germantown, N.Y.) and allowed to acclimate for 7 days prior toinitiation of the experiment. Animals were housed in appropriateisolated caging with ad lib sterile rodent food and acidified water anda 12:12 light:dark cycle. Animals were randomized into treatment groupsprior to tumor inoculation based on body weight. Randomization wasconfirmed based on tumor size immediately prior to initiation oftreatment.

Tumors: Tumors were inoculated by trochar placement of fragments fromrapidly growing tumors on donor animals. The human colon cancer cellline, HT-29, was used to initiate subcutaneous xenograft tumors.Cultured cells were trypsinized, washed, counted and resuspended at 50million cells per mL normal growth media. Tumors were inoculated byinjection of 0.1 mL (5 million cells) at the back of the neck. Tumorswere allowed to grow to an average size of 100 mm³ prior to initiationof treatment.

Monitoring: All animals were observed daily for general well-beingthroughout the experiments. Animals were weighed prior to tumorinoculation and weekly thereafter. Tumors were measured twice weeklythroughout the experiment, beginning 5–10 days after tumor inoculation.Any animal observed to have 15% or greater weight loss from the initialstarting weight and any animal observed to have greater than 4,000 mm³tumor volume were excluded from the study.

Example 1 Preparation of Liposomes with Entrapped TopoisomeraseInhibitor

Liposomes were prepared and loaded with a selected topoisomeraseinhibitor as follows.

A. Liposome Preparation

The lipids hydrogenated soy phosphaticylcholine (HSPC), cholesterol(Chol) and mPEG-DSPE (at a ratio of 56.4:38.3:5.3 mol/mol) weredissolved in ethanol at 65° C. in a 250 mL round bottom. The lipids wereagitated continuously for at least 30 minutes at 65° C. The total lipidconcentration in ethanol solution was 3.7 g total lipid per 10 mLethanol.

The dissolved lipid solution was transferred to another 250 mL roundbottom flask containing 100 mL of 250 mM ammonium sulfate solutionequilibrated to 65° C. The ethanol:lipid:ammonium sulfate hydrationmixture was mixed continuously for at least one hour while maintainingthe temperature using a 65° C. water bath to form oligolamellar ethanolhydration liposomes.

The oligolamellar liposomes were size reduced using a Lipex thermobarrelextruder to pass the hydration mixture through polycarbonate membraneswith known pore size dimensions. The mixture was passed 5 times througha 0.20 μm pore diameter membrane, followed by 10 passes through a 0.10μm pore diameter membrane. The extruded liposomes contained ammoniumsulfate within the interior aqueous compartment(s) of the liposomes, aswell as in the exterior aqueous bulk phase medium in which they aresuspended. The sized liposomes were stored in the refrigerator untildiafiltration preceding the remote loading procedure.

100 mg of a selected topoisomerase inhibitor, MPE-camptothecin, CKD-602or topotecan, was dissolved in 40 mL 10% sucrose solution to yield aconcentration of 2.5 mg/mL. After dissolution, the solution was passedthrough a 0.20 μm filter to remove insoluble particulates.

B. Remote Loading of Liposomes

Ammonium sulfate and ethanol were removed from the external bulk aqueousphase immediately prior to remote loading by hollow fiber tangentialflow diafiltration with a 100 KDa nominal molecular weight cutoffcartridge. Constant feed volume was maintained, and at least sevenexchange volumes were used resulting in liposomes suspended in anexterior aqueous phase comprised of 10% sucrose.

After diafiltration, the liposomes were mixed with a selected drugsolution at a ratio (drug solution:liposomes) of 1:4 (vol/vol) andrapidly warmed to 65° C. using a pre-equilibrated jacketed vesselcontaining water. The temperature of the mixture was maintained at 65°C. for 40 to 60 minutes, after which the mixture was rapidly cooled inan ice-water bath. After remote loading, a sample of the liposomes wastaken to check for the presence of crystals, to determine percentencapsulation and to measure the mean particle diameter.

Unencapsulated drug was removed from the bulk phase medium by hollowfiber tangential flow diafiltration using a 100 kDa nominal molecularweight cutoff cartridge. At least eight exchange volumes were used,resulting in liposomally encapsulated drug suspended in an externalaqueous phase comprised of 10% sucrose 10 millimolar Histidine pH 6.5.

The final liposome preparation was sterile filtered using a 0.22 μmcellulose acetate syringe filter and stored refrigerated and protectedfrom light until use.

C. Characterization of Liposomes

Percent encapsulation was determined using size exclusion chromatographyto compare the percent drug in the void volume (liposomal encapsulated)to the total drug (void volume plus included volume). Drug concentrationin the column fractions was determined by absorbance. Mean particlediameter was determined using quasielectric laser light scattering(QELS). The total lipid concentration was assayed at the post-sterilefiltration stage in order to determine the drug to lipid ratio.Liposomes loaded with MPE-camptothecin, topotecan and7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin (CKD-602) were preparedand characterized. The results are shown in the table below.

liposome- entrapped MPE- liposome-entrapped liposome-entrapped Parametercamptothecin Topotecan CKD-602 lot no. 221AZ43A 221AZ43B 221AZ53 TotalLipid 17.81 μmol/mL 15.97 μmol/mL 14.079 μmol/mL concentration Drugconcentration 2.69 mg/mL 1.72 mg/mL 1.77 mg/mL (4.55 μmol/mL) (3.76μmol/mL) (3.77 μmol/mL) drug:lipid ratio 0.26 0.24 0.27 (mol/mol)(1:3.92) (1:4.25) (1:3.73) Mean Particle 99 nm 95.4 nm 96.7 nm diameterPercent Encapsulation 96.4% 99.9% 95.3%

Example 2 In Vivo Efficacy of Liposome-Entrapped MPE-Camptothecin

Liposomes containing entrapped MPE-camptothecin were prepared asdescribed in Example 1. The liposome entrapped drug and the free drugwere diluted in 5% dextrose in water as required to achieve the desiredconcentrations.

Nude mice were inoculated with the human colon cancer cell line HT-29 asdescribed above in the methods section. Seventy mice were randomized toone of seven treatment groups as follows: free drug at 24 mg/kg, 15mg/kg or 6 mg/kg; liposome entrapped drug at 24 mg/kg, 15 mg/kg or 6mg/kg; saline. Treatment was initiated when average tumor volume wasapproximately 75 mm³ on day 10 post-tumor inoculation. All treatmentswere administered as intravenous bolus injections given weekly for 3treatments, specifically on days 10, 16 and 23.

Tumor size during and following each experiment was used as the primaryevaluation of therapeutic efficacy. Body weight was evaluated to assesstoxicity. All tumor bearing animals were observed following cessation oftreatment, until euthanized based on criteria above. Experiments wereconcluded when a majority of control tumors achieved the maximal allowedvolume (4,000 mm³).

Tumor size in each animal was measured repeatedly at various timepoints, thus these measurements were regarded as correlated information.Since the tumor sizes over time after treatment were of interest,repeated measurement analyses was done for each data set. By examiningthe data, a log transformation seemed reasonable. Let Y denote theoriginal tumor measurement, let Z=log(Y+1). After transforming data,repeated measurement analyses was done for the transformed data Z. TheSAS procedure PROC MIXED was used. The log growth rate for eachtreatment group was calculated and used to compare the differenttreatment groups. Statistical significance was declared at the 0.05level, but due to multiple comparisons, adjustment to the type I errorwere done and a P-value of <0.0033 indicated a statistically significantdifference in any designated comparison.

The results are summarized in Tables 1 and 2 and in FIGS. 2A–2B.

Example 3 Dose Finding Study for Liposome-Entrapped MPE-Camptothecin

Liposomes containing entrapped MPE-camptothecin were prepared asdescribed in Example 1. The liposome entrapped drug and the free drugwere diluted in 5% dextrose in water as required to achieve the desiredconcentrations.

Nude mice were inoculated with the human colon cancer cell line HT-29 asdescribed above in the methods section. Seventy mice were randomized toone of seven treatment groups as follows: liposome entrapped drug at 0.1mg/kg, 0.5 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg or 20 mg/kg; and saline.Treatment was initiated when average tumor volume was approximately 75mm³ on day 9 post-tumor inoculation. All treatments were administered asintravenous bolus injections given weekly for 3 treatments, specificallyon days 9, 16 and 23.

The tumor size was evaluated and analyzed as described in Example 2, andthe results are shown in Tables 3 and 4 and in FIGS. 3A–3B.

Example 4 In Vivo Efficacy of Liposome-Entrapped Topotecan

A. Liposome Preparation

Liposomes containing topotecan were prepared as follows.

The lipids distearoylphospatidylcholine (DSPC) and(N-(carbonyl-methoxypolyethylene glycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine) (mPEG-DSPE) werecombined at a molar ratio of 95:5 and dissolved in ethanol at 70° C.using continuous agitation. The lipid concentration in the ethanolsolution was 8.9 grams per 10 mL ethanol.

Dextran sulfate-ammonium salt was prepared by ion exchangechromatography using dextran sulfate sodium salt as the startingmaterial. A 100 mg/mL solution of dextran sulfate ammonium salt wasprepared by dissolving dextran sulfate sodium salt in water andadjusting the solution pH to 5 using ammonium hydroxide.

100 mL of dextran sulfate solution was heated to 70° C. and combinedwith the ethanol solution of lipid while mixing to form oligolamellarliposomes. The temperature of the oligolamellar ethanol hydrationliposome dispersion was maintained at 70° C. for one hour withcontinuous mixing.

The post hydration mixture was heated to 70 degrees and size reducedusing a Lipex thermobarrel extruder through a series of polycarbonatemembranes to arrive at a particle size near 100 nm mean particlediameter. Typically, the sequence involved 5 passes through an 0.2 μmpore diameter membrane, followed by 10 passes through an 0.1 μm porediameter membrane.

Unentrapped dextran sulfate polymer and remaining ethanol were removedfrom the external bulk aqueous phase immediately prior to the activedrug loading step with eight volume exchanges using 350 mM sodiumchloride solution, followed by eight volume exchanges using a 10%sucrose solution. The diafiltration cartridge employed had a specifiednominal molecular weight cutoff of 100,000 Daltons.

A solution of topotecan was prepared at a concentration of 2.5 mg/mL in10% sucrose. The drug solution and diafiltered liposomes were combinedat a volume ratio of 4:1, and the temperature of the resulting mixturewas raised to 70° C. and maintained with constant stirring for one hour.Active drug loading was terminated by rapidly cooling the post-loadingmixture using an ice water bath.

Unentrapped drug was removed by diafiltration employing a cartridgehaving nominal molecular weight cutoff of 100,000 Daltons. Typically,8–10 volume exchanges were employed using 10% sucrose 10 mM Histidine pH6.5 as the exchange buffer.

Drug concentration was adjusted to the final value by assaying forpotency with a uv-vis absorbance measurement and diluting accordingly.

The final process step involved sterile grade filtration employing a0.22 μm filter prior to filling vials.

B. Liposome Characterization

Percent encapsulation was determined using size exclusion chromatographyto determine the percent drug in the void volume (“liposomal drug”) tothe total amount recovered in both the included and void volumefractions. Drug concentration was monitored using uv-vis absorbancespectrophotometry. Mean particle diameter was determined usingquasielastic laser light scattering. Total lipid was determined usingphosphorous assay. The results are summarized in the table below.

Liposome-entrapped Parameter Topotecan total lipid 17.2 mg/mL total drug2.1 mg/mL drug:lipid ratio (mol:mol) 0.238 mean particle diameter 87.3nm percent encapsulation 98.8

C. In Vivo Pharmacokinetics and Efficacy

Seventy two mice were inoculated with HT-29 cancer cells as describedabove in the methods section. Nine days after tumor inoculation, theanimals were treated weekly with one of the following intravenoustreatments: saline; liposome-entrapped MPE-camptothecin 4 mg/kg; freetopotecan 25 mg/kg; liposome entrapped topotecan at drug dosages of 2mg/kg, 5 mg/kg or 8 mg/kg. All treatments were administered asintravenous bolus injections given weekly for 3 treatments, specificallyon days 9, 16 and 23.

The tumor size was evaluated and analyzed as described in Example 2, andthe results are shown in Tables 6 and 7 and in FIGS. 5A–5B.

Example 5 In Vivo Efficacy of Liposome-Entrapped CKD-602

A. Liposome Preparation and Characterization

Liposomes containing CKD-602 were prepared as described in Example 4,except using a drug solution of CKD-602. The liposomes werecharacterized as described in Example 4 and the results are summarizedbelow.

Liposome entrapped Parameter CKD-602 total lipid 12.5 mg/mL total drug2.07 mg/mL drug:lipid ratio (mol:mol) 0.315 mean particle diameter 92.8nm percent encapsulation 94.7

B. In Vivo Pharmacokinetics and Efficacy

Seventy two mice were inoculated with HT-29 cancer cells as describedabove in the methods section. Eleven days after tumor inoculation, theanimals were treated weekly with one of the following intravenoustreatments: saline, liposome-entrapped MPE-camptothecin 4 mg/kg; freeCKD602 20 mg/kg; liposome entrapped CKD602 at drug dosages of 1 mg/kg, 2mg/kg or 4 mg/kg.

All treatments were administered as intravenous bolus injections givenweekly for 3 treatments, specifically on days 11, 18 and 25.

The tumor size in each animal was measured twice weekly during the studyto evaluate therapeutic efficacy. Body weight of each animal wasmonitored twice weekly to assess toxicity of the formulations. Theresults are shown in Tables 8 and 9 and in FIGS. 7A–7B.

Although the invention has been described with respect to particularembodiments, it will be apparent to those skilled in the art thatvarious changes and modifications can be made without departing from theinvention.

1. A composition for administration of7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin, comprising liposomescomposed of at least about 20 mole percent of a vesicle-forming lipidderivatized with a hydrophilic polymer chain, said liposomes having aninside/outside ion gradient effective to retain the7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin within the liposomes;and entrapped in the liposomes, the7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin at a concentration ofat least about 0.20 μmole drug per mole lipid, wherein said liposomecomposition provides at least a four-fold increase in anti-tumoractivity over the anti-tumor activity of the drug in free-form.
 2. Thecomposition of claim 1, wherein the hydrophilic polymer ispolyethyleneglycol having a molecular weight between about 500 Daltonsand about 5,000 Daltons.
 3. The composition of claim 1, wherein theliposomes further comprise a vesicle-forming lipid having a phasetransition temperature above 37° C.
 4. The composition of claim 3,wherein the vesicle-forming lipid is selected from the group consistingof hydrogenated soy phosphatidylcholine, distearoylphosphatidylcholine,and sphingomyelin.
 5. The composition of claim 1, wherein the liposomesare comprised of between about 20–95 mole percent hydrogenated soyphosphatidylcholine and between about 1–20 mole percentdistearoylphosphatidylethanolamine derivatized with polyethyleneglycol.6. The composition of claim 1, wherein the liposomes are comprised of 95mole percent hydrogenated soy phosphatidylcholine and 5 mole percentdistearoylphosphatidylethanolamine derivatized with polyethyleneglycol.7. The composition of claim 1, wherein the liposomes further include apolyanionic polymer.
 8. The composition of claim 7, wherein saidpolyanionic polymer is selected from the group consisting of dextransulfate, chondroitin sulfate A, polyvinylsulfuric acid, andpolyphosphoric acid.
 9. The composition of claim 1, wherein theanti-tumor activity is measured using an HT-29 tumor model.
 10. Acomposition for administration of7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin, comprising liposomescomposed of vesicle-forming lipids derivatized with a hydrophilicpolymer chain and having an inside/outside ion gradient effective toretain the 7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin within theliposomes; and entrapped in the liposomes, the7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin at a concentration ofat least about 0.20 μmole drug per μmole lipid, wherein said liposomecomposition provides at least a four-fold increase in anti-tumoractivity over the anti-tumor activity of the drug in free-form.
 11. Thecomposition of claim 10, wherein the hydrophilic polymer ispolyethyleneglycol having a molecular weight between about 500 Daltonsand about 5,000 Daltons.
 12. The composition of claim 10, wherein theliposomes further comprise a vesicle-forming lipid having a phasetransition temperature above 37° C.
 13. The composition of claim 12,wherein the vesicle-forming lipid is selected from the group consistingof hydrogenated soy phosphatidylcholine, distearoylphosphatidylcholineand sphingomyelin.
 14. The composition of claim 10, wherein theliposomes are comprised of 20–95 mole percent hydrogenated soyphosphatidylcholine and 1–20 mole percentdistearoylphosphatidylethanolamine derivatized with polyethyleneglycoland 5–60 mole percent cholesterol.
 15. The composition of claim 10,wherein the liposomes are comprised of 30–65 mole percent hydrogenatedsoy phosphatidylcholine, 5–20 mole percentdistearoylphosphatidylethanolamine derivatized with polyethyleneglycoland 30–50 mole percent cholesterol.
 16. The composition of claim 10,wherein the liposomes are comprised of 20–95 mole percentdistearoylphosphatidycholine and 1–20 mole percentdistearoylphosphatidylethanolamine derivatized with polyethyleneglycol.17. The composition of claim 10, wherein the liposomes further include apolyanionic polymer.
 18. The composition of claim 17, wherein saidpolyanionic polymer is selected from the group consisting of dextransulfate, chondroitin sulfate A, polyvinylsulfuric acid, andpolyphosphoric acid.
 19. The composition of claim 10, wherein theanti-tumor activity is measured using an HT-29 tumor model.
 20. Acomposition for treating a tumor in a subject, comprising liposomescomposed of a vesicle-forming lipid and between about 1–20 mole percentof a vesicle-forming lipid derivatized with a hydrophilic polymer, saidliposomes being formed under conditions that distribute the polymer onboth sides of the liposomes' bilayer membranes; and entrapped in theliposomes, a topoisomerase inhibitor at a concentration of at leastabout 0.10 μmole topoisomerase inhibitor per μmole lipid, wherein saidliposomes have an inside/outside ion gradient sufficient to retain thetopoisomerase inhibitor within the liposomes at the specifiedconcentration prior to in vivo administration, and wherein saidliposome-entrapped topoisomerase inhibitor has a longer bloodcirculation lifetime than the topoisomerase inhibitor in free form, andwherein the topoisomerase inhibitor is an ionizable camptothecinderivative, wherein said liposome composition provides at least afour-fold increase in anti-tumor activity over the anti-tumor activityof the drug in free-form.
 21. A composition according to claim 20,wherein the hydrophilic polymer is polyethyleneglycol having a molecularweight between 500 and 5,000 daltons.
 22. A composition according toclaim 20, wherein the liposomes include a vesicle-forming lipid having aphase transition temperature above 37° C.
 23. A composition according toclaim 20, which comprises a vesicle-forming lipid selected from thegroup consisting of hydrogenated soy phosphatidylcholine, distearoylphosphatidylcholine, and sphingomyelin.
 24. A composition according toclaim 20, wherein the liposomes are composed of 20–95 mole percentdistearoyl phosphatidyl choline and 1–20 mole percent distearoylphosphatidylethanolamine derivatized with polyethyleneglycol.
 25. Acomposition according to claim 20, wherein the camptothecin derivativeis selected from the group consisting of 9-aminocamptothecin,7-ethylcamptothecin, 10-hydroxycamptothecin, 9-nitrocamptothecin,10,11-methylenedioxycamptothecin, 9-chloro-10,11-methylenedioxycamptothecin, irinotecan,7-(4-methylpiperazinomethylene)-10,11 -ethylenedioxy-20(S)-camptothecin(MPE-camptothecin), 7-(4-methylpiperazinomethylene)-10,11-methylenedioxy-20(S)-camptothecin,9-dimethyl-aminomethyl-10-hydroxycamptothecin (topotecan) and7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin (CKD-602).
 26. Acomposition according to claim 20, wherein the liposomes include apolyanionic polymer within the liposomes, said polymer capable offorming a complex with said topoisomerase inhibitor.
 27. A compositionaccording to claim 26, wherein said polyanionic polymer is selected fromdextran sulfate, chondroitin sulfate A, polyvinylsulfuric acid, andpolyphosphoric acid.
 28. A composition for administration of atopoisomerase inhibitor, comprising liposomes composed ofvesicle-forming lipids and having an inside/outside ion gradienteffective to retain the drug within the liposomes; and entrapped in theliposomes, a topoisomerase inhibitor which is an ionizable camptothecinderivative at a concentration of at least about 0.20 μmole topoisomeraseinhibitor per μmole lipid, wherein said liposome composition provides atleast a four-fold increase in anti-tumor activity over the anti-tumoractivity of the drug in free-form.
 29. A composition according to claim28, wherein the camptothecin derivative is selected from the groupconsisting of 9-aminocamptothecin, 7-ethylcamptothecin,10-hydroxycamptothecin, 9-nitrocamptothecin,10,11-methylenedioxycamptothecin, 9-chloro-10,11-methylenedioxycamptothecin, irinotecan,7-(4-methylpiperazinomethylene)-10,11 -ethylenedioxy-20(S)-camptothecin(MPE-camptothecin), 7-(4-methylpiperazinomethylene)-10,11-methylenedioxy-20(S)-camptothecin,9-dimethyl-aminomethyl-10-hydroxycamptothecin (topotecan) and7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin (CKD-602).
 30. Acomposition according to claim 28, wherein the liposomes include apolyanionic polymer within the liposomes, said polymer capable offorming a complex with said topoisomerase inhibitor.
 31. A compositionaccording to claim 28, wherein said polyanionic polymer is selected fromdextran sulfate, chondroitin sulfate A, polyvinylsulfuric acid, andpolyphosphoric acid.